Project Summary/Abstract Telomeres are specialized nucleoprotein structures at the ends of eukaryotic chromosomes that are required for chromosome stability and cellular proliferation. These structures are essential for human health because dysregulation of either telomere protection or telomerase activity causes many inherited and acquired human diseases, with telomere dysfunction also closely tied to cancer and aging. Chromosomal ends consist of tandem repeats of TG-rich sequences that terminate in a highly conserved 3 single-stranded DNA (ssDNA) overhang. Management of this single-stranded overhang is one of the most critical aspects of telomere maintenance. When left unprotected, this overhang initiates DNA damage responses, leading to catastrophic events that permanently damage the genome and result in apoptosis or senescence. Recent data point to the intricate integration of the general DNA-maintenance and telomere machineries. Understanding the interplay of the genomic and telomere-specific ssDNA-binding factors is key to understanding the basic biology of chromosome maintenance and the catastrophic consequences of its misregulation. This program is focused on two protein complexes that manage ssDNA in many chromosomal contexts, including at telomeres and replication forks. Human CTC1/STN1/TEN1(hCST) is a heterotrimeric protein complex that protects and maintains sites of G-rich ssDNA throughout the genome. It acts prominently at telomeres, binding the conserved G-rich overhang to coordinate the termination of telomerase activity and recruitment of C-strand fill in by DNA pola-primase. Mutants of hCST are associated with a range of human diseases characteristic of proliferation defects. As a first step in understanding the mechanism of action of hCST, we have solved the high-resolution structure of hCST bound to ssDNA using cryoEM. This structure provides surprising insights into hCST function and is an excellent starting point to address key mechanistic questions regarding its function, such as its interaction with ssDNA, addressed in Aim 1, and the importance of the decamer structure in cells, investigated in Aim 2. Our structure reveals that hCST most strikingly resembles replication protein A (RPA), also a heterotrimeric complex involved in the non-specific binding of ssDNA during replication, repair and recombination. The driving hypothesis for Aim 3 is based on the observations that, in the context of certain mutants, RPA and CST activities can substitute for one another. This leads to the testable hypothesis that these RPA mutants reveal a cryptic G-specific binding activity contained within the protein. The structural, functional and biochemical parallels between the CST and RPA complexes suggest a highly tuned interplay of their activities that allow for their crosstalk in the management of difficult G-rich regions of chromatin, and this will be addressed in this aim.